The Tomato Feruloyl Transferase FHT Promoter Is an Accurate Identifier of Early Development and Stress-Induced Suberization

As a wall polymer, suberin has a multifaceted role in plant development and stress responses. It is deposited between the plasma membrane and the primary cell wall in specialized tissues such as root exodermis, endodermis, phellem, and seed coats. It is formed de novo in response to stresses such as wounding, salt injury, drought, and pathogen attack and is a complex polyester mainly consisting of fatty acids, glycerol, and minor amounts of ferulic acid that are associated to a lignin-like polymer predominantly composed of ferulates. Metabolomic and transcriptomic studies have revealed that cell wall lignification precedes suberin deposition. The ferulic acid esterified to ω-hydroxy fatty acids, synthetized by the feruloyl transferase FHT (or ASFT), presumably plays a role in coupling both polymers, although the precise mechanism is not understood. Here, we use the promoter of tomato suberin feruloyl transferase (FHT/ASFT) fused to GUS (β-glucuronidase) to demonstrate that ferulate deposition agrees with the site of promoter FHT activation by using a combination of histochemical staining and UV microscopy. Hence, FHT promoter activation and alkali UV microscopy can be used to identify the precise localization of early suberizing cells rich in ferulic acid and can additionally be used as an efficient marker of early suberization events during plant development and stress responses. This line can be used in the future as a tool to identify emerging suberization sites via ferulate deposition in tomato plants, which may contribute to germplasm screening in varietal improvement programs.


Introduction
The wall polymer suberin has a multidimensional role in plant development and stress responses [1,2]. Structurally, it is a glycerol-based, fatty-acid-derived polyester comprised primarily of ω-hydroxy acids, α, ω-dicarboxylic acids, fatty alcohols, and small amounts of hydroxycinnamic acids (mainly ferulic acid) [3]. It is deposited between the plasma membrane and the cell wall in specialized tissues, such as root exodermis, endodermis, seed coat, and phellem (cork), in root and aboveground tissue in woody plants [4]. Moreover, suberin can be formed de novo in response to stress such as wounding, salt injury, drought, and pathogen attack [5]. In addition to providing strength to the cell wall, suberin averts water loss and pathogen access by sealing off the layer of suberized cells. It acts as a potent barricade against pathogens, and in addition to providing strength to the cell wall, it may also act as an antimicrobial fence [6]. Recently, it has been shown that microbiota inhabiting the roots can also influence suberin deposition at the endodermis [7].

Figure 1.
Schematic representation of the enzymatic function of suberin feruloyl tr FHT catalyzes the conjugation of feruloyl-CoA to aliphatic chains such as ω-hyd primary alcohols to form feruloyl esters [19][20][21], potentially acting as a linker o nin-like or other cell wall polymers.
Here, we present a method that allows precise localization of early sub using the promoter of tomato suberin feruloyl transferase (FHT) f (β-glucuronidase). The aim of this study is to determine whether the site moter activation correspond to those of ferulate deposition and thus can robust marker for early suberization events. Transgenic tomato plan ProSlFHT::GUS showed ferulate deposition at the position of promoter ind ized using alkali UV microscopy and in accordance with the predicted SlF This tool can be used as an efficient marker of early suberization event development and stress responses. Such markers will aid in the funda standing of the suberization process and germplasm screening for varietal programs in tomato against diverse stress responses.

FHT Proteins of Different Plant Species have a Conserved HxxxD Motif Invol and a DFGWG Motif Located at the C-Terminal End
In agreement with the characteristics of BAHD acyltransferases, all F different plant species have a conserved HxxxD motif involved in ca DFGWG motif located at the C-terminal end, the latter of which is presu structural function [21]. Protein homologs of tomato FHT gene (Solyc03 used for amino acid sequence alignment in ClustalW and visualized by M BAR (http://bar.utoronto.ca/, accessed on 2 May 2023) webpage ( Figure 2) analysis showed that the tomato FHT gene is very close in ancestry to th (StFHT) gene (PGSC0003DMG400031731), which has been characterized in [28]. FHT catalyzes the conjugation of feruloyl-CoA to aliphatic chains such as ω-hydroxy acids and primary alcohols to form feruloyl esters [19][20][21], potentially acting as a linker of suberin to lignin-like or other cell wall polymers.
Here, we present a method that allows precise localization of early suberization sites using the promoter of tomato suberin feruloyl transferase (FHT) fused to GUS (βglucuronidase). The aim of this study is to determine whether the sites of FHT promoter activation correspond to those of ferulate deposition and thus can function as a robust marker for early suberization events. Transgenic tomato plants expressing Pro SlFHT ::GUS showed ferulate deposition at the position of promoter induction, visualized using alkali UV microscopy and in accordance with the predicted SlFHT functions. This tool can be used as an efficient marker of early suberization events during plant development and stress responses. Such markers will aid in the fundamental understanding of the suberization process and germplasm screening for varietal improvement programs in tomato against diverse stress responses.

FHT Proteins of Different Plant Species Have a Conserved HxxxD Motif Involved in Catalysis and a DFGWG Motif Located at the C-Terminal End
In agreement with the characteristics of BAHD acyltransferases, all FHT proteins of different plant species have a conserved HxxxD motif involved in catalysis and a DFGWG motif located at the C-terminal end, the latter of which is presumed to have a structural function [21]. Protein homologs of tomato FHT gene (Solyc03g097500) were used for amino acid sequence alignment in ClustalW and visualized by Mview using the BAR (http: //bar.utoronto.ca/, accessed on 2 May 2023) webpage ( Figure 2). Phylogenetic analysis showed that the tomato FHT gene is very close in ancestry to the potato FHT (StFHT) gene (PGSC0003DMG400031731), which has been characterized in deeper detail [28].

Putative Cis Elements Found in Tomato FHT Promoter
The putative SlFHT promoter region (1710 bp upstream of the translation initiation) was examined using the PLACE [31] and the PlantCare [32] databases [29]. In agreement with the reported function of the FHT gene as a key acyltransferase involved in suberin biosynthesis, sequence analysis showed the presence of cis-regulatory motifs specific to abiotic stresses such as wounding, salt injury, water stress, etc. (e.g., WBOXNTERF3) and biotic stresses such as pathogenesis and salicylic acid receptiveness (e.g., GT1GMSCAM4, WBOXATNPR1). As expected, a number of ABA responsive motifs were present (e.g., WB-BOXPCWRKY1, MYB and MYC binding sites, SORLIP1, and WRKY), since suberin biosynthesis is known to be regulated by this hormone [33][34][35]. In addition, motifs corresponding to organ-/cell-/and tissue-specific activation of phenylpropanoid genes (e.g., EBOXBN-NAPA) and root and seed inducible motifs were present (e.g., ROOTMOTIFTAPOX1, RYREPEATBNNAPA) (Table 1)  Protein homologs of tomato FHT gene (Solyc03g097500) were obtained from www.phytozome.jgi.doe.gov, accessed on 2 May 2023, and matches with more than 85% similarity were used for amino acid sequence alignment in ClustalW and visualized by Mview using BAR (http://bar.utoronto.ca/, accessed on 2 May 2023) webpage. In agreement with the characteristics of BAHD acyltransferases, all FHT proteins of different plant species have conserved HxxxD motif involved in catalysis and DFGWG motif located at the C-terminal end, the latter of which is presumed to have a structural function [21].

Induction of Pro SlFHT :GUS in Tomato Tissues Undergoing Developmental Suberization
We hypothesized that the tomato FHT promoter (Pro SlFHT ) could be used as a good marker to study early suberization at the sites of suberin nucleation in different tissues. Hence, we generated Pro SlFHT ::GUS lines in tomato in order to elucidate the participation of tomato FHT promoter in tissues known to deposit suberin. For this, we used the tomato variety Hawaii 7996 (H7996), which has been shown to induce ligno-suberin vascular coating to restrict infection of the vascular pathogen Ralstonia solanacearum [28].
A fragment consisting of 1713 bp upstream of the initial ATG codon of SlFHT was amplified from genomic DNA and fused to the reporter β-glucuronidase (GUS) to generate the Pro SlFHT ::GUS construct, which was transformed into H7996 tomato. In agreement with the critical role reported for FHT/ASFT/RWP in suberization [19][20][21], we clearly observed the induction of Pro SlFHT ::GUS in tissues known to accumulate suberin. Tomato root exodermal cells are known to deposit suberin [36]. We observed strong induction of Pro SlFHT ::GUS in the root outer layers of young seedlings ( Figure 3A). Further, prior to emergence, we observed strong induction of Pro SlFHT ::GUS in lateral root primordia, indicating a hardening process by suberin deposition in the cells of the developing lateral root cap ( Figure 3B). To analyze whether the induction of Pro SlFHT ::GUS in tomato tissues corresponded to an increase in ferulates, in accordance to the function of FHT, we used a technique whereby ferulates can be detected by emission of blue fluorescence with UV excitation at neutral pH that characteristically changes to a stronger green emission under conditions of high pH such as in the presence of alkali [37][38][39]. We observed that the UV autofluorescence detected in root epidermal cells and lateral root primordia changed from blue to a strong green color upon treatment with alkali (1N KOH pH above 10) ( Figure 3C,D), indicating the accumulation of ferulates in these tissues ( Figure 3C,D). As expected, this pattern was highly coincidental to the observed induction of Pro SlFHT ::GUS, highlighting the robustness of this method to report dual ferulate/FHT promoter activation.

Induction of ProSlFHT:GUS in Tomato Tissues Undergoing Developmental Suberization
We hypothesized that the tomato FHT promoter (ProSlFHT) could be used as a good marker to study early suberization at the sites of suberin nucleation in different tissues. Hence, we generated ProSlFHT::GUS lines in tomato in order to elucidate the participation of tomato FHT promoter in tissues known to deposit suberin. For this, we used the tomato variety Hawaii 7996 (H7996), which has been shown to induce ligno-suberin vascular coating to restrict infection of the vascular pathogen Ralstonia solanacearum [28].
A fragment consisting of 1713 bp upstream of the initial ATG codon of SlFHT was amplified from genomic DNA and fused to the reporter β-glucuronidase (GUS) to generate the ProSlFHT::GUS construct, which was transformed into H7996 tomato. In agreement with the critical role reported for FHT/ASFT/RWP in suberization [19][20][21], we clearly observed the induction of ProSlFHT::GUS in tissues known to accumulate suberin. Tomato root exodermal cells are known to deposit suberin [36]. We observed strong induction of ProSlFHT::GUS in the root outer layers of young seedlings ( Figure 3A). Further, prior to emergence, we observed strong induction of ProSlFHT::GUS in lateral root primordia, indicating a hardening process by suberin deposition in the cells of the developing lateral root cap ( Figure 3B). To analyze whether the induction of ProSlFHT::GUS in tomato tissues corresponded to an increase in ferulates, in accordance to the function of FHT, we used a technique whereby ferulates can be detected by emission of blue fluorescence with UV excitation at neutral pH that characteristically changes to a stronger green emission under conditions of high pH such as in the presence of alkali [37][38][39]. We observed that the UV autofluorescence detected in root epidermal cells and lateral root primordia changed from blue to a strong green color upon treatment with alkali (1N KOH pH above 10) ( Figure 3C,D), indicating the accumulation of ferulates in these tissues ( Figure 3C,D). As expected, this pattern was highly coincidental to the observed induction of ProSlFHT::GUS, highlighting the robustness of this method to report dual ferulate/FHT promoter activation.

Induction of Pro SlFHT :GUS in Tomato Tissues Undergoing Wound Healing
Since suberin deposition is known to occur as part of the wound-healing response [29,40], we analyzed the induction of Pro SlFHT ::GUS upon injury. At 48 h post pin-prick injury on leaves, a strong induction of Pro SlFHT ::GUS was observed surrounding the injured region ( Figure 4A,B). Further, when water imbalance or other factors lead to fruit cracks in tomato, the plants have a mechanism to seal this crack to prevent rotting due to the growth of saprophytes. We observed specific induction of Pro SlFHT ::GUS in the sealing region of the cracks, indicating suberization in this particular wound-healing response ( Figure 4C,D). Further, we visualized ferulate deposition using alkali UV microscopy concomitantly with FHT promoter activation (GUS signal) during pin-prick injury and fruit cracks in tomato ( Figure 4E,F).

Induction of ProSlFHT:GUS in Tomato Tissues Undergoing Wound Healing
Since suberin deposition is known to occur as part of the wound-healing response [29,40], we analyzed the induction of ProSlFHT::GUS upon injury. At 48 h post pin-prick injury on leaves, a strong induction of ProSlFHT::GUS was observed surrounding the injured region ( Figure 4A,B). Further, when water imbalance or other factors lead to fruit cracks in tomato, the plants have a mechanism to seal this crack to prevent rotting due to the growth of saprophytes. We observed specific induction of ProSlFHT::GUS in the sealing region of the cracks, indicating suberization in this particular wound-healing response ( Figure 4C,D). Further, we visualized ferulate deposition using alkali UV microscopy concomitantly with FHT promoter activation (GUS signal) during pin-prick injury and fruit cracks in tomato ( Figure 4E,F).

Induction of Pro SlFHT ::GUS in Vascular Suberization Response in Tomato against Pathogens
Suberin vascular coating in response to the vascular pathogen Ralstonia solanacearum infection has been observed in the resistant tomato cultivar H7996 [28]. Pro SlFHT ::GUS transgenic H7996 tomato plants were inoculated through their roots by soaking the soil with R. solanacearum with a concentration of~1 × 10 7 CFU mL −1 and grown at 28 • C for 20 days. In water-treated plants, induction of Pro SLFHT ::GUS was not observed in the xylem vasculature ( Figure 5A). In infected resistant tomato plants, induction of Pro SlFHT ::GUS was observed in the xylem vascular tissue as well as in the outer layers of the root ( Figure 5B). Interestingly, we observed that the UV autofluorescence detected in vascular coatings in response to R. solanacearum infection in resistant H7996 ( Figure 5D) changed from blue to a strong green color upon treatment with alkali (1N KOH pH above 10)-but not in response to mock treatment ( Figure 5C)-indicating the presence of ferulates in these coatings ( Figure 5F,G), as previously reported [28]. In contrast, no such vascular coating was observed in water-treated plants (non-infected) ( Figure 5E,G). These data indicate that resistance to R. solanacearum in H7996 is mediated by induction of the FHT expression in the vasculature, which is accompanied by ferulate deposition in the same area. The lipophyllic fluorescent dye fluorol yellow, which stains the aliphatic portion of suberin, was also used in Pro SLFHT ::GUS transgenic tomato plants infected with R. solanacearum and mock-treated plants. As shown in Figure S1, infection with R. solanacearum resulted in a clear increase in root fluorescence compared to mock-treated roots. However, this dye provided less zonal information regarding potential sites of R. solancearum-induced suberin deposition in tomato roots.

Induction of ProSlFHT::GUS in Vascular Suberization Response in Tomato against Pathogens
Suberin vascular coating in response to the vascular pathogen Ralstonia solanacearum infection has been observed in the resistant tomato cultivar H7996 [28]. ProSlFHT::GUS transgenic H7996 tomato plants were inoculated through their roots by soaking the soil with R. solanacearum with a concentration of ~1 × 10 7 CFU mL −1 and grown at 28 °C for 20 days. In water-treated plants, induction of ProSLFHT::GUS was not observed in the xylem vasculature ( Figure 5A). In infected resistant tomato plants, induction of ProSlFHT::GUS was observed in the xylem vascular tissue as well as in the outer layers of the root ( Figure  5B). Interestingly, we observed that the UV autofluorescence detected in vascular coatings in response to R. solanacearum infection in resistant H7996 ( Figure 5D) changed from blue to a strong green color upon treatment with alkali (1N KOH pH above 10)-but not in response to mock treatment ( Figure 5C)-indicating the presence of ferulates in these coatings ( Figure 5F,G), as previously reported [28]. In contrast, no such vascular coating was observed in water-treated plants (non-infected) ( Figure 5E,G). These data indicate that resistance to R. solanacearum in H7996 is mediated by induction of the FHT expression in the vasculature, which is accompanied by ferulate deposition in the same area. The lipophyllic fluorescent dye fluorol yellow, which stains the aliphatic portion of suberin, was also used in ProSLFHT::GUS transgenic tomato plants infected with R. solanacearum and mock-treated plants. As shown in Figure S1, infection with R. solanacearum resulted in a clear increase in root fluorescence compared to mock-treated roots. However, this dye provided less zonal information regarding potential sites of R. solancearum-induced suberin deposition in tomato roots.

Discussion
Suberin deposition as a response to multiple stresses has recently been explored with renewed vigor to develop plants resilient to abiotic and biotic stresses [2]. In tomato, suberin plays vital roles against diverse environmental stresses [41]. However, the absence of markers or tools for localizing suberizing tissues remains as an impediment for the fundamental understanding of the process in tomato and other crop plants, particularly during the response to various stresses, as well as during germplasm screening for varietal improvement programs. In tomato, the visualization of suberin in tissues mostly relies on stains such as Sudan IV or fluorol yellow, which target the aliphatic components [42] and cannot capture the early accumulation of ferulate esters catalyzed by FHT, which is expressed earlier than the fatty acyl biosynthetic machinery [43,44] and before suberin lamellae structuring. Suberin undergoes enormous compositional changes as the lamella matures. In the initial stages, lignin-like polymer is laid down in advance to the suberin polyester [45]. In fact, ferulates constitute a crucial component of the lignin-like polymer, potentially acting as nucleating sites for suberin matrix polymerization [29,46]. Hence, our tool using the promoter of SlFHT fused to GUS allows precise localization of early suberization sites. FHT is a key acyltransferase, which catalyzes the conjugation of feruloyl-CoA to aliphatic chains to form feruloyl esters, which are suberin precursors [19][20][21]29].
The putative SlFHT promoter region showed presence of cis-regulatory motifs specific to abiotic stresses such as wounding, salt injury, water stress, etc. (e.g., WBOXNTERF3) and to biotic stresses such as pathogenesis and salicylic acid receptiveness (e.g., GT1GMSCAM4, WBOXATNPR1), and as expected, a number of ABA responsive motifs were present (e.g., WBBOXPCWRKY1, MYB and MYC binding sites, SORLIP1, and WRKY) since suberin biosynthesis is known to be regulated by ABA [33][34][35]. In addition, motifs corresponding to tissue-specific activation of phenylpropanoid genes (e.g., EBOXBNNAPA) and root and seed inducible motifs were present (e.g., ROOTMOTIFTAPOX1, RYREPEATBNNAPA). The tomato FHT gene is very close in ancestry to the potato FHT gene [28], and its promoter was also reported to contain similar cis-regulatory motifs [29]. In agreement with the critical role reported for FHT in suberization [17,18,24], we observed the induction of Pro SlFHT ::GUS in the outer root layers, which could correspond to root exodermis, which is well known to deposit suberin [36]. Further, we observed strong induction of Pro SlFHT ::GUS in the lateral root primordia, indicating a hardening process by suberin-ferulate deposition in the cells of the developing lateral root cap before emergence. Interestingly, the root caps of lateral roots have been reported to contain a cuticle-like polymer in Arabidopsis [47]. In contrast, we observed that lateral root primordia initiated suberization during their development in tomatoes. It is possible that both lipid-based polymers, suberin and cutin, having common ancestry [48], perform similar functions towards root cap hardening. However, ferulic acid, which is a chief component of suberin-associated phenolics, is a minor component in the chemically similar cutin polymer [1]. In accordance with the function of FHT, we observed ferulate deposition using alkali UV microscopy at the sites of promoter activation. Since suberin deposition is known to occur as part of the woundhealing response [29,40], we also observed a strong induction of Pro SlFHT ::GUS surrounding the tissues at 48 h post pin-prick injury on leaves. Further, when water imbalance or other factors lead to fruit cracks in tomato, the plants have a mechanism to seal this crack to prevent rotting due to the growth of saprophytes. We could observe specific induction of Pro SlFHT ::GUS in the sealing region of the cracks, indicating suberization in this particular wound-healing response. In all cases, a positive correlation between FHT promoter activity and ferulate deposition by alkali UV microscopy was observed. Additionally, induction of Pro SlFHT ::GUS in the vasculature of resistant tomato cultivar H7996 against pathogen R. solanacearum was observed. Suberin vascular coating in response to vascular pathogen R. solanacearum infection has been previously reported in resistant tomato cultivar H7996 [28]. In infected resistant tomato plants, induction of Pro SlFHT ::GUS was observed in the xylem vascular tissue, and ferulate vascular reinforcements were also observed, showing positive correlation between induction of Pro SlFHT ::GUS and ferulate deposition after infection with R. solanacearum.
Together, these data indicate that this tool can be used as an efficient marker of early suberization events during plant development and stress responses. It can potentially be translated to other vascular pathogens to tomato as well as for studying other stress responses such as salt injury, drought stress, etc. Further, the tomato FHT promoter is a good candidate for tissue-specific expression of desired genes and metabolic engineering at targeted sites such as the exodermis, lateral root primordia, wound-healing zones, and other sites undergoing suberization to confer resilience to diverse stresses. In conclusion, we have developed a very effective tool to detect the sites for early suberization events linked to FHT activation and ferulate deposition.

Plant Material and Growth Conditions
The tomato (Solanum lycopersicum) variety Hawaii 7996 (H7996) was used for all experiments. Seeds were germinated and plants were grown in pots containing soil (Substrate 2, Klasmann-Deilmann GmbH) mixed with perlite and vermiculite (30:1:1) in controlled growth chambers at 60% humidity and 12 h photoperiod with light intensity of 120-150 µmol·m −2 ·s −1 . The temperature was set at 27 • C when using LED lighting and at 25 • C when using fluorescent lighting.

DNA Constructs
The Pro FHT ::GUS construct was generated using the Gateway system (Invitrogen, Waltham, MA, USA). A fragment consisting of 1713 bp upstream of the initial ATG codon (Solyc03g097500) was amplified from genomic DNA of H7996 tomato using the forward primer (ProFHTF1: ACAAGTTTGTACAAAAAAGCAGGCTAAACAACAAAATAAGATTG-CAC) and the reverse primer (ProFHTR1: ACCACTTTGTACAAGAAAGCTGGGTTTTCT-CAAAATTAATAAATCCTG) containing the attB flanking sequences. This sequence was cloned into the Gateway entry vector pDONR 207 using a BP reaction and then transferred into the Gateway destination vector pGWB3 using an LR reaction.

Stable Transformation of Tomato
Pro FHT ::GUS construct was transformed into H7996 using Agrobacterium tumefaciens strain C58C1. A. tumefaciens was used for co-culture with tomato cotyledons. Explant preparation, selection, and regeneration were performed following standard protocols [49]. Transformants were selected on kanamycin-containing MS medium and propagated into subsequent generations.

Detection of SlFHT Promoter Activity
Plant tissues or taproot-hypocotyl transition zone sections were immersed in an icechilled 90% (v/v) acetone bath and incubated for 20 min on ice, after which they were rinsed with water. Tissues were vacuum-infiltrated for 20 min with a solution containing 1 mM 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-Gluc), 50 mM sodium phosphate buffer (pH 7), 1 mM potassium ferrocyanide, 1 mM potassium ferricyanide, and 0.05% (v/v) Triton X-100. Samples were then incubated at 37 • C for a maximum of 48 h. Stained tissues were washed 2-3 times with phosphate-buffered saline (PBS) and then cleared with 70% (v/v) ethanol and stored in 70% (v/v) ethanol. Images were obtained using an Olympus SZX16 stereomicroscope equipped with a DP71 camera system.

Histological Methods
Thin tangential sections were made with a sterile razor blade of leaf discs, fruit section, or taproot-hypocotyl transition zone for histological assays. Autofluorescence from ferulates bound to the cell wall shows a pH-dependent blue-to-green color conversion [37][38][39]. Ferulates in the xylem vascular tissue were visualized by mounting cross-sections in 70% ethanol (neutral pH) and illuminating them with UV with excitation bandpass filter in the range 340-380 nm to observe blue autofluorescence. These same sections were subsequently mounted in 1N KOH (pH above 10) to observe green autofluorescence from ferulates. Green color intensity calculation was performed with ImageJ software by selecting the vascular areas around the main vessels with localized fluorescence or green signal.

Bacterial Inoculation in Plants
Four-to five-week-old tomato plants were inoculated through their roots with Ralstonia solanacearum GMI1000 using the soil drenching method [50]. For this, roots were wounded by making four holes in the soil at the corners of the pot with a 1 mL pipette tip and inoculated with a 1 × 10 7 CFU mL −1 (OD 600 = 0.01) suspension of bacteria [50]. Inoculated plants were kept in a growth chamber at 28 • C for 20 days.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author (N.S.C.), upon reasonable request.